Clotting of blood is a complicated process involving a large number of blood components including fibrinogen and prothrombin which is converted to thrombin. It has long been recognized that many aspects of unexplained bleeding or abnormal clotting can be explained in terms of improper levels of these materials in the blood. For instance, states of hypo-fibrinogenemia or hyper-fibrinogenemia may result from hepatic disease, from disseminated intravascular coagulation, from fibrinolytic syndrome, neoplastic disease, and post-operatively due to trauma. By monitoring the fibrinogen, thrombin and prothrombin levels within the blood, a physician may acquire meaningful data concerning the patient's blood clotting abilities. For example, the Activated Partial Thromboplastin Time (APTT) Test measures coagulation factors of the intrinsic pathway. These factors include Factors XII, XI, IX, VIII, X, V, II and I which may be abnormal based on heredity or heparin therapy. Thus, the APTT test is useful as a presurgical screen and for monitoring heparin therapy. Similarly, fibrinogen testing (by the Thrombin Time (TT) test or quantitative fibrinogen test) provides useful diagnostic data when unexplained bleeding or abnormal clotting occurs.
As a result, substantial efforts have been made to measure these clotting components, particularly that of fibrinogen, the most difficult of these to measure accurately. Most methodologies rely upon immunologic and clotting techniques although clearly the latter is preferred. The immunologic techniques, although generally capable of precisely defining the levels of the various components within the blood stream, are incapable of distinguishing between active and inactive forms. Accordingly, the immunologic methods are felt to be less accurate with respect to the patient's actual clotting ability.
Consequently, the results obtained by clotting techniques are preferred as being more clinically significant. Most of these methods rely upon the addition of excess thrombin to dilute plasma and the measurement of the resultant clotting time is then related to the fibrinogen concentration of the plasma. This is the original fibrinogen assay described by Clauss in Gerinnungsphysiologische Schelimethode Zur Bestimung Des Fibrigens, ACTA Haemat 17:237-246 (1957).
Another useful reference regarding the processes and components involved in blood coagulation and methods for monitoring such coagulation are disclosed in "Hemostatsis and Thrombosis, A Conceptual Approach", Churchill, Livington, U.S. 1979.
Typically, most instruments detect the formation of a clot by monitoring either optical turbidity or electrical conductivity. The latter represents the traditional approach employed by the so-called fibrometer-type of instrument. Effectively, this instrument measures increasing conductivity which may be correlated to the formation of clots. Similarly, turbidity may be optically sensed by the decrease in light transmission due to the formation of a clot. Certainly with the normal PT or APTT tests, these methods have found widespread acceptance despite the fact that each test has associated therewith a level of indefiniteness regarding the point at which the clot is determined to have occurred. Inasmuch as the fibrometer represents the traditional approach, and most physicians and clinicians are accustomed to utilizing this approach, as a practical matter all other instruments, to be accepted, should have a high degree of correlation with the fibrometer.
It is one aspect of the present invention to provide improved methods especially useful with optical clot detection techniques which have a high level of correlation with the standard fibrometer.
Detection of fibrinogen levels has historically been the most difficult of the tests to perform particularly with hypo-fibrinogenemic samples. This occurs because the formation of the clot is a lengthy process subject to substantial error in the determination of when that clot has formed. Substantial problems are incurred with the need to discriminate between true clot formation and aberrant signal noise accruing as a result of reagent mixing, and the passage of air bubbles or other nonrelevant particulate matter in front of the optical sensors. Often, these noise producers may be erroneously interpreted by the instrument as early clots and a false early clot detection displayed. This occurs as a result of the enormous difficulty associated with determining what incoming data represents a clot as opposed to aberrant noise. This problem is characteristic of the forward looking approach characteristic of conventional instruments which analyze data as it is accumulated.
It is an object of the present invention to provide new and improved methods useful for fibrinogen and quantitative fibrinogen detection.
It is a related object to provide methods which may be used for other coagulation monitoring including PT and APTT.
It is yet another aspect of the present invention to provide methods useful for optical clot detection. These and other objects and aspects of the present invention will become clear upon study of the ensueing detailed description.